Objective To explore the application of particle-gun technique in transfecting dendritic cells(DCs),and investigate the expression of DNA vaccine particle IgHV1-GM-CSF in DCs induced by particle-gun.Methods Monocytes were isolated from donor peripheral blood collected by Ficoll density gradient method and adherent culture,and then differentiated into immature DCs(imDCs) by rhGM-CSF and rhlL-4 induction.The plasmids for transfection were IgHV1-GM-CSF/pcDNA3.0 and pEGFP,which were prepared with endofree plasmid purification kit.The DCs were transfected with plasmid pEGFP by particle-gun at different transfection conditions,the green fluorescence positive cell and the total cell numbers were counted respectively under fluorescence microscope,and the transfection efficiency was calculated;the viable cell count was performed after trypan blue staining;the transfection efficiencies of particle-gun,liposome-mediated transfection and electroporation method were compared.The plasmids IgHV1-GM-CSF/pcDNA3.0 and pEGFP were cotransfected into imDCs by particle-gun under the optimum conditions,and then DNA was extracted,the expression of IgHV1-GM-CSF was determined by RT-PCR,and of GM-CSF by ELISA.Results The optimum conditions of particle-gun transfection in imDCs were: gas pressure at 120psi,PVP concentration in 0.02mg/ml,microcarrier loading in 0.5mg gold/shot,and DNA loading ratio in 1.5?g plasmid/mg gold.The transfection efficiencies of particle-gun and electroporation were 10.5%?2.4%(n=3) and 45.2%?5.6%(n=3) respectively,while that of liposome-mediated transfection was very low,and significant difference existed among the three methods(P

Objective To investigate the effect of ligustrazine on the expression of platelet-derived growth factor-?(PDGF-?) receptor and extracellular signal regulated kinase(ERK1/2) induced by angiotensin Ⅱ(AngⅡ) in cardiac myocytes,and explore the mechanism of therapeutic.Methods Cultured cardiac myocytes of neonatal rats were treated with 10-7 mol/L AngⅡ as Ang Ⅱ group,10-7 mol/L AngⅡ plus 10 mg/L ligustrazine as ligustrazine group,the normally cultured neonatal rat cardiac myocytes as control group.Protein synthesis was measured by -leucine incorporation,and the expression of PDGF-? receptor and ERK1/2 was detected by Western blot.SPSS 11.0 software was used to analyze the data.Results There were significant differences among 3 groups(F=20.71 P

Objective To determine the antigen-specific CTL in PBMC induced by a fusional family-gene vaccine of the immunoglobulin heavy chain variable gene framework region combined with the sequence of cytokine CM-CSF in vitro with MHC pentamer. Methods Peripheral blood samples were collected from two healthy donors and two patients. One was follicular lymphoma and another was hair cell leukemia. PBMC were isolated by density gradient centrifugalization with Ficoll and then subsequently differentiated into immature DCs (imDCs) induced by recombinant human GM-CSF and recombinant human IL-4. Gene gun was used to deliver the plasmids of the gene vaccine or the control plasmids into the imDCs. RT-PCR and ELISA assay were used to detect IgHVl-GM-CSF mRNA and GM-CSF in order to validate the transfection of the vaccine. After adding the cytokine cocktail, the imDCs became mature DCs. Then the mature DCs were co-cultured with lymphocytes from the blood samples for the induction of the antigen-specific CTL. The cultured cells were classified into vaccine group and control group and harvested at different time points of 0 d,7d, 17 d and 24 d after transfection. The subset of CD3+CD8+ T cells was analyzed by FCM assay. Finally, the CTL levels were detected with fluorescently labeled MHC pentamer antibody targeting vaccine epitopes. Results With the induction of cytokines, the imDCs with typical morphology were generated in PBMC. After delivering, the efficient expressions of the vaccine in the imDCs were determined by RT-PCR. And ELISA results also confirmed that GM-CSF was produced at a level of (28 ±6) ng/106 cells of the imDCs loaded with the vaccine, which was significantly different from that of control group (10 ± 3) ng/106 cells (t = 5. 191, P <0.01). FCM assay result showed that the CD3+ CD8+ T cells increased in a stepwise pattern during the culture. For control group, the levels at 0 d,7d, 17d and 24 d were ( 34. 24 ± 2. 72 )% , (46.06 ± 3.08)%, ( 65. 34 ± 4. 26 )% and (73.86 ±4.85 )% , respectively. For vaccine group, the results were (32. 28 ± 2. 08 ) % , (45. 32 ± 3. 81)% , ( 63. 37 ± 4. 21)% and (75. 01 ±3. 20)%. The differences between each time point had statistical significance (F = 176. 966 ,P <0.01) ,but there was no statistical differences between vaccine group and control group ( F = 0.657,P>0.05). The MHC pentamer analysis showed that the DCs loaded with IgHV1-GM-CSF fusional vaccine could efficiently induce the antigen-specific CTL response and the CTL levels increased gradually with the culture time, with the highest level of 4. 36% in the lymphoma blood and 3. 89% in the hair cell leukemia blood. Conclusions MHC pentamer assay could efficiently determine the antigen-specific CTLs response induced by the gene vaccine of family IgHV frame region in vitro. It could be a useful method for monitoring of anti-tumor cell immunity and evaluating of diagnosis and prognosis of the tumors in clinical application.

Norovirus ; chemistry ; Receptors, Virus ; physiology ; Viral Structural Proteins ; chemistry ; immunology ; Viral Vaccines ; immunology

Adult ; Carcinoid Tumor ; pathology ; Ear Neoplasms ; pathology ; Ear, Middle ; pathology ; Female ; Humans

Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Breast ; pathology ; Breast Neoplasms ; drug therapy ; pathology ; Cyclophosphamide ; therapeutic use ; Diagnosis, Differential ; Doxorubicin ; therapeutic use ; Female ; Follow-Up Studies ; Humans ; Lymphoma, B-Cell ; drug therapy ; pathology ; Lymphoma, Large B-Cell, Diffuse ; drug therapy ; pathology ; Male ; Middle Aged ; Prednisone ; therapeutic use ; Sarcoma ; pathology ; Spleen ; pathology ; Splenic Neoplasms ; drug therapy ; pathology ; Vincristine ; therapeutic use

Aim To explore the effect of shikonin on stemness maintance of glioma stem cells ( GSCs ). Methods After the U87-MG cells were cultured and isolated, the sphere cells were identified by immuno-fluorescent staining. The alteration of stemness of GSCs by shikonin treatment(2 μmol·L - 1 ) for 12 h, 24 h and 48 h was valued by morphological detection using optical microscope and sub-sphere forming assay. Mo-reover, the related markers of stem cells, such as CD133, were detected in shikonin treated GSCs by western blot assay. Protein expression of PI3K, p-PI3K, Akt and p-Akt was detected by western blot af-ter shikonin treatment alone. Furthermore, by combi-nation with insulin-like growth factor-1 ( IGF-1), we observed the alteration of stemness maintance of shiko-nin-treated GSCs. Results The presence of neural stem cell related markers CD133 and nestin proved the characteristics of GSCs. Shikonin treatment significant-ly inhibited the morphology of GSCs and the sub-sphere forming. Besides, the reduced expression of CD133 was detected in shikonin treated GSCs. Though, the expression of PI3K and Akt did not change compared with the control group, the expression of p-PI3K and p-Akt was reduced. Furthermore, the combination of IGF-1 markedly attenuated the inhibitory effect of shikonin on stemness maintance of GSCs. Conclusion The stemness maintance of GSCs can be significantly inhibited by shikonin treatment, in which PI3K/ Akt pathway is involved.

OBJECTIVETo characterize the morphological features of thyroid papillary microcarcinoma (PMC) and assess the significance of expression of CK19, HBME-1, Galectin-3, CD56 and p63 in differential diagnosis of PMC from benign thyroid lesions.

METHODSClinicopathologic features of 78 cases PMC were reviewed. Immunohistochemical analysis of CK19, HBME-1, Galectin-3, CD56, and p63 in 78 cases of PMC and 48 cases of benign thyroid lesions (18 cases of papillary hyperplasia, 17 cases of nodular goiter and 13 cases of lymphocytic thyroiditis) was conducted. The patients were followed up for from 6 to 269 months after surgical operation.

RESULTS69 cases nuclear atypia and overlapping nuclei (88.5%), 67 cases nuclear grooves (85.9%), 50 cases nuclear pseudoinclusions (64.1%) and 60 cases papillary architecture (76.9%) were detected in 78 cases of PMC. Moderate to strong co-expression of CK19, HBME-1 and galectin-3 was observed in 98.0% (50/51) in the PMC group but in none of the benign disease group. The expression of CD56 and p63 was negative in both groups. In the postoperative follow-up period of 6-269 months, 7 cases (9.0%) developed intrathyroid recurrence, 3 cases (3.8%) developed lymph node metastasis, no distant metastasis or death was observed. In 12 cases (15.4%) the PMC lesion smaller than 3 mm in diameter was not found by frozen section diagnosis.

CONCLUSIONSOverlapping nuclei, nuclear atypia, polar disorder, ground glass nuclei, nuclear grooves and nuclear pseudoinclusions are most important for the diagnosis of PMC with or without papillary architecture. The appearance of definite interstitial invasion, interstitial sclerosis and true complex papillary architecture are more helpful to make right diagnosis. Intraoperative frozen section is of limited value for a reliable diagnosis of PMC in diameter < or = 3 mm. Moderate to strong co-expression of CK19, HBME-1 and Galectin-3 is a very useful indicator for differential diagnosis of PMC from benign thyroid lesions.

Adult ; Biomarkers, Tumor ; metabolism ; CD56 Antigen ; metabolism ; Carcinoma, Papillary ; diagnosis ; metabolism ; pathology ; surgery ; Cell Nucleus ; pathology ; Diagnosis, Differential ; Female ; Follow-Up Studies ; Galectin 3 ; metabolism ; Goiter, Nodular ; metabolism ; pathology ; Humans ; Hyperplasia ; Keratin-19 ; metabolism ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Recurrence, Local ; Thyroid Gland ; metabolism ; pathology ; Thyroid Neoplasms ; diagnosis ; metabolism ; pathology ; surgery ; Thyroidectomy ; methods ; Thyroiditis, Autoimmune ; metabolism ; pathology ; Transcription Factors ; metabolism ; Tumor Suppressor Proteins ; metabolism

Objective To investigate the diagnostic value of double balloon endoscopy (DBE) in the elderly. Methods Clinical manifestations and endoscopic findings of 42 elderly patients (aged 60-80 years) and 73 young and middle-aged patients (aged 12-59 years) with small bowel lesions were obtained and compared. Factors influencing the diagnostic outcome of DBE in patients with small bowel bleeding were identified,and the optimal check time after the latest bleeding was determined.Results The procedures of 85.7％ (36/42) in the elderly and 79.5％(58/73) in young and middle aged were completed (P＞0.05).There was no significant difference in the procedure time between the two age groups.No severe complications were observed in the elderly group.The overall positive rate by double balloon enteroscopy examination were 71.4 ％ (30/42) and 63.0 ％ (46/73),respectively in the two groups (P＞ 0.05). Ulcer and tumor lesions were the most common findings,and diverticula and angiodysplasia were the second common findings. Longer duration of bleeding and higher number of bleeding episodes were found in the elderly with positive DBE findings than those with negative findings. Positive diagnostic rate was significantly higher when DBE was performed within 7 days than that after 7 days (90％ vs. 40％). Conclusions DBE is a safe,reliable diagnostic modality,especially in the elderly patients with small bowel bleeding in which ulcer and tumor lesions are the most common identifications.DBE is of greater benefit in patients with more bleeding episodes over a long period,and should be performed within 7 days after the last bleeding.

Objectives To investigate and evaluate the preventive and therapeutic effects of celecox ib,a selective cyclooxygenase-2(COX-2)inhibitor,on multiple colorectal adenorna and compare it with aspirin.Methods Ninty-six patients with colorectal multiple adenoma were randomly divided into A,B and C groups.Adenomas in all patients were removed with high-frequency eleetrocoagulation,electroexci- sion or argon plasma coagulation(APC)under colonoscopy.Then,group A were administered celecoxib 200 mg twice daily,group B aspirin 50 mg twice daily,group C served as control.Colonoscopy was per formed every 6 months in the first year,and every year in order to observe and evaluate the recurrence rate of adenoma and the side effects after the treatment.Results Twenty-seven patients in group A,26 pa- tients in group B and 27 patients in group C had completed the treatment.At the end of the treatment, on PP/ITT analysis,the cure rate of the eolorectal adenoma were 84.4%/100% ,78.1%/96.2% and 75.0%/88.9% in group A,B and C,respectively.During the first year of follow-up,there were 1 ,1 and 6 cases which were found recurrences of the adenomas in group A,B and C,respectively.The recurrence rates of coloreetal adenomas in group A(3.7%)and group B(4.0%)were significantly low er than that in group C(24.0%) (P＜0.05 and＜0.05,respectively).At the end of follow-up,the total recurrence rate of colorectal adenomas in group A(14.80%)and group B(19.2%)were significant- ly lower than that in group C(46.2%)(P＜0.05 and＜0.05).While the side-effective rate regroup A (3.3%)was significantly lower than that in group B(22.5%)(P＜0.05).Conclusions After re- section of the multiple colorectal adenomas,both the selective inhibitor of COX-2,celeeoxib and the non- selective inhibitor of COX-2,aspirin,may reduce its recurrence rate,but the former has a good tolerance and lower side-effects.